Administration of RBC and particularly older RBC units is proposed to potentiate toxicity in patients receiving transfusions. Consistent with this concept, data from our institution show an adverse effect of aged (>2wk storage) leukocyte-depleted RBC in promoting tissue injury and mortality in trauma patients. The mechanisms underlying this so-called 'RBC-lesion' phenomenon remain unclear. Two proposed mechanisms include i) dysfunction in mechanisms by which RBC couple oxygen sensing by hemoglobin with the stimulation of nitric oxide (NO) signaling in the vasculature. In this context, 3 mechanisms have been forwarded, S-nitrosohemoglobin, nitrite-reduction and ATP. ii) stimulation of immune cells and subsequent exacerbation of inflammatory tissue injury. In this proposal we propose to test the importance of these two mechanisms by coupling clinical evaluation of microvascular hemodynamics and immune cell function with mechanism based studies to determine how ageing negatively impacts RBC function in these contexts. We hypothesize that Aged RBC have a depleted capacity to couple oxygen sensing to NO-bioactivity via defects in nitrite-reductase and ATP pathways, and an increased capacity to stimulate inflammation in the recipient. This hypothesis will be tested by pursuit of the following aims 1) Determine the effect of a single unit of leukocyte-depleted RBC on microcirculation hemodynamics and inflammation in resuscitated trauma patients, 2) Determine the role of banked RBC on the 3 proposed mechanisms of controlling hypoxic NO-signaling and 3) Determine the effects of banked RBC on innate immune activation and inflammation. We will evaluate mechanisms by which RBC of different ages mediate vascular NO-signaling and modulate immune-cell function both in vivo and in vitro in a paired fashion. The proposed studies will employ standard of care approaches with stable trauma patients in the ICU which will only be transfused if clinically indicated. We will restrict measurements to pre- and post administration of 1 unit of blood and hence one age and blood type (thereby avoiding potential confounding variables associated with administration of multiple units of blood comprising different ages etc). In addition, we will restrict the study to patients who receive blood in morning hours to allow assessment of mechanisms (aim 2 and aim 3). It is anticipated that completion of the proposed studies will provide key mechanistic insights into the nature of the stored RBC-lesion assessed both in vivo and ex-vivo. PUBLIC HEALTH RELEVANCE: Relevance Statement In this proposal we aim to elucidate how administration of packed Red blood cells (RBC) and particularly older stored RBC may contribute to toxicity in patients requiring transfusion. We will focus on two specific areas related to i) how RBC control blood flow via regulating the vasodilator nitric oxide and ii) how RBC modulate immune cell and complement function and subsequent inflammatory reactions. We anticipate that elucidation of these mechanisms will improve our understanding of RBC functions in transfusions and allow the design of therapies to limit any toxic effects.